1. Field of the Invention
The present invention relates to biocontrol of diseases caused by the soil-borne fungus Gaeumannomyces graminis. In particular, the invention relates to strains of fluorescent Pseudomonas species (spp.) which have unique root-colonizing ability for small grain crops and biocontrol activity for diseases caused by Gaeumannomyces graminis in small grain crops and take-all patch in turf grass. The invention further relates to isolation and identification of the unique strains, and application thereof to control plant diseases caused by Gaeumannomyces graminis. 
2. Description of the Art
Widespread diseases of small grain crops and turf grass are caused by the soil-borne fungus Gaeumannomyces graminis (Gg), and result in significant economic losses due to reductions in crop yield. Take-all, a disease caused by Gaeumannomyces graminis var. tritici (Ggt) occurs in all wheat-growing regions of the world and is the most important root disease of wheat. Symptoms of wheat take-all include dark longitudinal lesions on roots; in severe cases, the entire root may become blackened with disease with the fungus migrating to the crown of the wheat plant (where the crown roots originate) and the tillers (stems). Severely infected wheat plants are identified in the field by their white heads which result when infection of the crown by the fungus cuts off water transport to upper plant parts causing the plant to die prematurely. Yield losses can be considerable up to 50% of the potential wheat yield. There are no resistant wheat cultivars and registered fungicides perform inconsistently. Further, growers are being increasingly challenged to grow wheat with minimum or no tillage to reduce soil erosion. These practices increase the severity of take-all and other root diseases. Although wheat is particularly susceptible to the take-all fungus, many other Gramineae such as barley, rye, and triticale can also be infected.
Traditionally, take-all has been controlled by a combination of crop rotation and tillage, practices which reduce the inoculum potential of the pathogen. However, because long rotations are often not economically feasible and tillage contributes to soil erosion, the trend in cereal production is toward less tillage and two or three wheat crops before a break. Both of these practices exacerbate take-all. There is no known source of genetic resistance in wheat against take-all, and methods of chemical control are limited. The need for agriculture to become more sustainable and less dependent on chemical pesticides has necessitated the development of alternative approaches to control take-all and other soil-borne diseases.
Other Gg fungi, for example, Gaeumannomyces graminis var. avenae (Gga) infects oats and grasses and have been identified as causing take-all patch in turf grasses such as bent grass. Gaeumannomyces graminis var. graminis (Ggg) infects some grasses and has been suggested as causing crown sheath rot in rice.
All agricultural soils show some degree of antagonism to Ggt and other soil-borne pathogens. This has been referred to as xe2x80x9cgeneral suppressionxe2x80x9d (N. Gerlagh, Netherlands Journal of Plant Pathology 74:(Suppl. 2) 1-97 (1968) or xe2x80x9cgeneral antagonismxe2x80x9d (D. Hornby, Annual Review of Phytopathology, Annual Reviews Inc. Palo Alto, Calif. (1983), pp. 65-85). General antagonism results from the overall microbial activity in a soil. In addition, a xe2x80x9cspecificxe2x80x9d suppression (biological control) of Ggt (known as take-all decline) develops in certain circumstances which is superimposed over xe2x80x9cgeneral suppressionxe2x80x9d and which results in a nearly complete control of take-all. Take-all decline (TAD) is a natural biological control of take-all, defined as the spontaneous reduction in disease and the increase in yield with extended monoculture of Ggt-susceptible small grain crops such as wheat and barley. TAD was first observed more than 50 years ago and is now recognized as a worldwide phenomenon. The similarity of TAD throughout the world is remarkable in view of the broad range of soil types, climates, and agronomic conditions under which wheat, barley, and other small grains are cultivated. Field studies have clearly indicated that the development of TAD follows a consistent pattern everywhere, requiring the continuous cultivation of a small grain and the presence of the take-all pathogen. Factors such as soil type and previous cropping history only seem to modulate the extent and speed of development of TAD. Despite the fact that take-all eventually declines, most growers abandon monculture prematurely because interim losses can be considerable. Once established, however, TAD permits a recovery in yield and persists as long as monoculture continues. Practical exploitation of TAD offers the potential as a natural biological control of take-all. However, to do this, the responsible mechanism(s) for TAD would need to be identified and applied. However, research to date has been mostly descriptive and no particular occurrence of TAD is yet fully understood. A similar decline of take-all patch caused by Gga occurs in established turf.
TAD has been extensively studied in an attempt to determine the mechanisms responsible for natural take-all suppression. The most common theories put forward to explain this phenomenon include changes in the microbiological status of the soil, build up of antagonistic bacteria, changes in the pathogenicity and population of the fungus, and presence of protective fungi (D. Hornby in xe2x80x9cTake-All Decline: A Theorists""s Paradise,xe2x80x9d Soil-borne Plant Pathogens, Ed. B. Schippers and W. Gams, Academic Press, New York (1979), pp. 133-156 and D. Hornby, Annual Review of Phytopathology, Annual Reviews Inc., Palo Alto, Calif. (1983), pp. 65-85). Homby reviewed these explanations and concluded that no single mechanism could explain TAD worldwide, and this view has been universally accepted by those working in the field of disease suppressive soils.
The most widely held explanation for TAD is based on microbial interactions between the take-all pathogen and specific antagonistic root-associated microorganisms (R. J. Cook and A. D. Rovira, Soil Biology and Biochemistry 8: 269-273 (1976)). Several types of evidence support a role for microbial antagonism in the suppression of Ggt. For example, suppressiveness can be transferred by incorporation of a small amount (1-10% w/w) of a TAD (suppressive) soil into a take-all conducive soil. Furthermore, the suppressiveness of a TAD soil is eliminated by pasteurization of the soil with moist heat (60xc2x0 C., 30 min.), by soil fumigation with methyl bromide or by growing crops which are non-hosts of the pathogen.
Studies of the microbial antagonism involved in TAD have focused on attempts to identify specific Ggt-antagonistic microorganisms and to transfer these organisms to soil to reproduce suppression. A wide variety of microorganisms have been tested given the prevailing idea that the specific strains responsible differ among TAD soils. Cook and Rovira, 1976, supra, originally hypothesized that among the antagonistic microorganisms the fluorescent Pseudomonas spp. have a key role in TAD. U.S. Pat. No. 4,456,684 describes Pseudomonas strains which suppress diseases caused by take-all and other Gg fungi and methods for selection and application of the strains.
Many of the most effective strains produced the antibiotic 2,4-diacetylphloroglucinol (Phl) (C. Keel et al., Applied and Environmental Microbiology 62:552-563 (1996)). Phl is a phenolic metabolite with activity against a variety of bacteria, viruses, and fungi, including the take-all pathogen (reviewed in L. S. Thomashow and D. M. Weller, In: G. Stacey and N. T. Keen (eds.) Plant-microbe Interactions, Vol. I, Chapman and Hall, Ltd. London, pp. 187-236 (1996)). J. M. Raaijmakers et al. (Applied and Environmental Microbiology 63:881-887 (1997)) report that Phl-producing fluorescent Pseudomonas spp. were present on roots of wheat grown in three TAD soils from Washington State (USA). In take-all conducive soils collected from sites near the TAD fields, Phl-producing fluorescent Pseudomonas. spp. were not detected or were detected at densities at least 40-fold lower than those in the TAD soils. Although use of microbial biocontrol agents holds great promise as a practical means to control soilborne pathogens, all published or patented biocontrol agents for take-all have the disadvantages of performing inconsistency, being soil-specific, and being unable to duplicate the level of control consistently observed in a TAD soil. No microorganism tested to date has demonstrated the disease control abilities expected of a strain involved in TAD. Thus, it is not surprising that no biocontrol agent for take-all has been commercialized. What is needed are effective biocontrol agents for take-all which duplicate the suppressiveness of a TAD soil and are effective independent of soil type, and perform consistently.
We have discovered unique biocontrol agents for control of the diseases caused by the soil-borne fungus Gaeumannomyces graminis (Gg) in small grain crops and turf grass. The invention encompasses unique strains of fluorescent Pseudomonas species (spp.) which suppress (inhibit the incidence of or reduce the incidence or severity of) diseases caused by Gg, such as take-all, at low doses, and have root-colonizing ability which is greater than any previous biocontrol agent of Gg. In addition, the root colonization ability and biocontrol activity of the strains are not affected by soil type.
The biocontrol agents of the invention provide biocontrol which is consistently greater than that of all known biocontrol agents for diseases caused by Gg, such as take-all. Additionally, the strains have the further unique property of being able to duplicate the level of biocontrol observed in a TAD soil.
The biocontrol agents of the invention comprise biologically pure cultures of strains of fluorescent Pseudomonas spp. which contain a biosynthetic locus which encodes for the production of the antibiotic 2,4-diacetylphloroglucinol, which have a unique genotype as shown by a characteristic unique Random Amplified Polymorphic DNA (RAPD) profile, exhibit biocontrol activity at dose levels 10 to 1000 times lower than take-all-suppressive microorganisms known heretofore, and exhibit superior root colonizing ability as demonstrated by 10 to 1000-fold higher population density and extended colonizing activity. We have found that exemplary strains of the invention have the ability to colonize roots at a population density averaging at least 105 colony forming units per gram of root, including the rhizosphere soil, for at least 7 successive growth cycles. Such root-colonizing ability is unprecedented. Further, the novel strains are not affected by soil type.
The invention further comprises methods of isolation and identification of these unique strains. A protocol for screening of bacteria is shown in FIG. 2, and the screening method is described in detail, below.
A further aspect of the invention is application of the unique strains or compositions comprising the strains for biocontrol of plant diseases caused by Gaeumannomyces graminis. When used as a seed, soil, furrow treatment or drench, the unique strains of the invention have the ability to suppress Gg under field conditions. Application of the strains to seed or soil showed the unprecedented duplication of suppression of take-all equivalent to natural take-all decline (see Table 1, below), which has never been shown before. Further, because the strains suppress take-all at low dose levels, the strain can be grown and applied at a cost of nearly 10 to 1000-fold less than other currently existing biocontrol agent of take-all. In addition, because the strains are responsible for natural TAD, it is envisioned that they need only be applied only once in a field. All other biocontrol agents of take-all require repeated application.
In accordance with this discovery, it is an object of the invention to provide unique strains of fluorescent Pseudomonas spp. which provide biocontrol of the diseases caused by Gg which is greater than known biocontrol strains.
It is also an object of the invention to provide biocontrol strains that have root colonizing ability (higher root population density and extended activity) which is greater than any known biocontrol agent for take-all. This property is particularly valuable because it is known that for suppression of root diseases such as take-all, increases in root colonization result in greater and more consistent biocontrol activity.
Another object of the invention is the provision of biocontrol agents for control of diseases caused by Gaeumannomyces graminis in small grains and turf grass which are not affected by soil type.
It is a further object of the invention to provide methods based on RAPD analysis for selecting the strains of the invention having a characteristic banding pattern.
A still further object of the invention is the provision of methods for biologically controlling take-all in small grain crops and patch in turf grass using the strains of the invention and agricultural compositions which incorporate the strains.
Other objects and advantages of the invention will become readily apparent from the ensuing description.